White/Opaque switching in the human fungal pathogen Candida albicans is an alternation between two distinct types of cells, White and Opaque. Each state is heritable for many generations, and switching between states occurs stochastically, at low frequency. This interconversion, which plays important roles in both pathogenesis and mating, exemplifies two characteristics shared by many examples of cell differentiation: The conversion from one cellular state to another is stochastic, and each state, once formed, is heritable for many generations. This proposal seeks to determine the molecular machinery that underlies White/Opaque switching as well as the heritability of the two states. The results have implications for the stochastic nature of cell differentiation as well as the ability of C. albicans to cause disease in humans. The first specific aim seeks to understand why and how the four major regulators of White/Opaque switching work together in what appears to be a higher order complex, and how this complex effects heritable maintenance of the Opaque cell transcriptional program. Affinity purification and mass spectrometry will be used to identify proteins which associate with Wor1, the master regulator of White/Opaque switching. Genetic analysis will be used to confirm the roll of these Wor1-associated proteins in regulating the White/Opaque switch, while appropriate biochemical techniques will be used to characterize the function of the Wor1-associated complex. The second aim is to establish a hierarchical as well as a temporal order to White/Opaque switching that will illuminate (1) why switching appears to be all or none and (2) why each state, once formed, is heritable for many generations. This will be accomplished by: monitoring genome- wide transcript levels throughout the process of coordinated switching, whole-genome chromatin immunoprecipitation assays, and targeted deletion of specific regulatory genes and transcriptionfactor binding sites. Taken together, the experiments outlined in these aims should lead to the development of a comprehensive model of the mechanism of White/Opaque switching, the properties that underlie its heritability, and the structure of the large circuit (over 400 genes) controlled by this switch in C. albicans. Candida albicans is a major human fungal pathogen which causes a wide array of diseases, ranging from "common yeast infections" to life-threatening systemic infections. PUBLIC HEALTH RELEVANCE: The research proposed in this application seeks to describe a unique aspect of C. albicans biology that is linked to the ability of this organism to cause disease and provides an attractive target for the development of novel anti-fungal therapeutics.